Davids B J, Wu X J, Yoshino T P
University of Wisconsin-Madison, School of Veterinary Medicine, Department of Pathobiological Sciences, Madison, WI 53706, USA.
Gene. 1999 Mar 4;228(1-2):213-23. doi: 10.1016/s0378-1119(99)00008-6.
A cDNA encoding an integrin subunit was cloned and structurally characterized from an embryonic cell line derived from Biomphalaria glabrata, snail intermediate host of the human blood fluke Schistosoma mansoni. Cells of the B. glabrata embryonic (Bge) snail cell line were initially tested for their sensitivity to the integrin-specific tetrapeptide inhibitor Arg-Gly-Asp-Ser (RGDS). Washed Bge cells when exposed to 0.5 to 2.0mM of RGDS were significantly inhibited in their ability to spread on a glass substrate. Spreading inhibition was specific, since a control peptide Arg-Gly-Glu-Ser (RGES) did not have the same effect. RT-PCR was performed using previously reported degenerate oligonucleotide primers to the ligand binding domain (LBD) of known beta integrin subunits and Bge cDNA. A 137 bp fragment was amplified, TA-cloned, sequenced, and the na and deduced aa sequences were compared with other beta integrins. Databank analysis showed that the 137 bp product shared >/=55.6% aa similarity to other beta integrin LBDs. Southern and northern blot analyses using the 137 bp sequence as a probe revealed binding to Bge genomic DNA restriction fragments and to an approximately 8 kb poly-(A)+RNA transcript, respectively. An exact 5' primer synthesized to the 137 bp product and an oligo-d(T) primer then were used to amplify from Bge cDNA, a partial beta integrin sequence of 2285 bp that contained a 1971 bp ORF. The remaining upstream coding region was obtained using 5' RACE methods. The complete ORF, consisting of 2364 bp, encoded a 788 aa sequence with shared similarity to other known beta integrins (44.6-61.5%). Sequence and structural comparisons, which include a characteristic LBD, a series of three homologous cysteine-rich repeats, membrane proximal sequence (LLTFIHD), cytoplasmic NPXY motifs, and predicted domain lengths of the molluscan protein, clearly identifies it as an integrin homologue. This report represents the first cloning of a cDNA putatively encoding an integrin subunit from molluscan cells, and establishes the Bge cell line as a model for studying cellular adhesion in molluscs at the molecular level.
从曼氏血吸虫的中间宿主——光滑双脐螺的胚胎细胞系中克隆出一个编码整合素亚基的cDNA，并对其进行了结构表征。最初检测了光滑双脐螺胚胎（Bge）细胞系的细胞对整合素特异性四肽抑制剂Arg-Gly-Asp-Ser（RGDS）的敏感性。当洗涤后的Bge细胞暴露于0.5至2.0mM的RGDS时，其在玻璃基质上的铺展能力受到显著抑制。铺展抑制具有特异性，因为对照肽Arg-Gly-Glu-Ser（RGES）没有相同的效果。使用先前报道的针对已知β整合素亚基配体结合域（LBD）的简并寡核苷酸引物和Bge cDNA进行RT-PCR。扩增出一个137bp的片段，进行TA克隆、测序，并将核苷酸和推导的氨基酸序列与其他β整合素进行比较。数据库分析表明，137bp的产物与其他β整合素LBD具有≥55.6%的氨基酸相似性。使用137bp序列作为探针进行Southern和Northern印迹分析，分别揭示了与Bge基因组DNA限制片段和大约8kb的聚腺苷酸加尾RNA转录本的结合。然后，合成一个与137bp产物精确匹配的5'引物和一个寡聚d(T)引物，用于从Bge cDNA中扩增出一个2285bp的部分β整合素序列，该序列包含一个1971bp的开放阅读框（ORF）。使用5' RACE方法获得其余的上游编码区。由2364bp组成的完整ORF编码一个788个氨基酸的序列，与其他已知的β整合素具有共同的相似性（44.6 - 61.5%）。序列和结构比较包括一个特征性的LBD、一系列三个同源的富含半胱氨酸的重复序列、膜近端序列（LLTFIHD）、细胞质NPXY基序以及软体动物蛋白的预测结构域长度，清楚地将其鉴定为整合素同源物。本报告代表了首次从软体动物细胞中克隆出一个推测编码整合素亚基的cDNA，并将Bge细胞系确立为在分子水平上研究软体动物细胞粘附的模型。
